There has been only a modest progress in implementing effective means for the treatment of patients with glioblastoma multiforme (GBM). We have previously uncovered a plasma membrane receptor that is over- expressed in around 80% of patients with GBM and not normal brain, interleukin 13 receptor alpha 2 (IL- 13Ra2). This receptor has served the development of targeted cytotoxins, viruses and vaccines, and remains an attractive target for other molecular means to diagnose, image and treat GBM. We have recently generated three different agents that recognize IL-13Ra2 through various ways that have potential to be further developed for imaging and treatment of the disease. First, a heptapeptide was selected from the phage peptide display library that binds specifically IL-13Ra2. This peptide has two important features: (i) it binds the receptor independent of the native ligand, and (ii) it induces receptor's internalization in preliminary experiments. Second, a doubly-targeted agent was designed and produced, which is composed of receptor-recognition unit (IL-13K, a mutated form of native ligand), a portion of a bacterial toxin that enables cytosolic transport (D2) and a nuclear localization signal (NLS);IL-13.E13K.D2.NLS. This recombinant protein recognizes cells over-expressing IL-13Ra2 and according to its design's purpose, delivers the C-terminal portion to the cells'nuclei. This opens a novel way of delivering drugs/labels not only to a targeted subpopulation of cells, but also directly to their preferable/desirable site of cellular action. Third, six novel monoclonal antibodies (MAb) were generated that recognize specifically IL-13Ra2 in both humans and dogs. Dogs develop spontaneous malignant gliomas and their clinical and pathological and molecular features, including over-expression of IL-13Ra2, resemble human disease. Also, recent clinical studies with MAb in brain tumors demonstrated efficacy and the availability of several of them against an attractive target in GBM will allow a careful examination of this not well-explored approach in the treatment of GBM. Thus, the proposal has three Specific Aims. Aim #1 is to examine pre-clinically newly selected peptides that bind IL-13Ra2 for specific recognition and targeting of GBM. The nature of the peptide's binding to the receptor will be explored in more detail as well as its utility for tumor imaging, and more specifically, for the imaging of tumor responses to therapies. Aim #2 is to develop novel ligands of unique multi-level targeting capacity: specific recognition of GBM cells and concomitant targeting of micro-organelles of the same cells. In this aim, recombinant proteins will be developed for delivering Auger electron emitters directly to GBM cells nuclei. In Specific Aim #3, newly generated monoclonal antibodies against IL-13Ra2 will be further analyzed and develop into highly specific GBM targeting agents. Recombinant designer forms of some of these antibodies will be produced and tested for anti-tumor efficacy. The results of this project will permit further successful clinical application of various targeted peptides/proteins as anti-GBM drugs and/or imaging agents. PUBLIC HEALTH RELEVANCE: Most malignant tumors of the brain, including glioblastoma multiforme (GBM) remain incurable and thus represent unmet need in medicine. We have identified three novel separate means to target specifically GBM tumor cells, and also their vulnerable internal compartments, that can potentially complement each other and/or verify each other's effectiveness in the diagnosis and treatment of GBM. It is expected that these new means will increase the armamentarium of available agents controlling the progression and/or recurrence of the disease.